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MOLECULAR BIOLOGY > PREFI LEC: GENE MUTATIONS > Flashcards

PREFI LEC: GENE MUTATIONS Flashcards

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1
Q

Permanent alteration in the DNA sequence that makes up a gene

A

GENE MUTATION

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2
Q

Causes of GENE MUTATION?

A

errors in DNA replication, exposure to various environmental factors (radiation, chemicals, or certain viruses)

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3
Q

Wide range of effects GENE MUTATION?

A

harmless, genetic disorders, development of diseases (cancer)

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4
Q

substitution of 1 nucleotide, will not change the amino acid sequence

A

Silent

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5
Q

change the amino acid sequence, but the replacement & the original amino acid have similar biochemical properties

A

Conservative

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6
Q

replacement of amino acid w/ a biochemically different amino acid

A

Nonconservative

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7
Q

terminates proteins prematurely when a nucleotide substitution produces a stop codon

A

Nonsense

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8
Q

addition/deletion of nucleotides in a DNA sequence disrupts the reading frame

A

Frameshift

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9
Q

purine replaces a purine or pyrimidine with a pyrimidine

A

Transition

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10
Q

purine replaces a pyrimidine or pyrimidine with a purine

A

Transversion

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11
Q

 Used to directly analyze the change in protein structure/function  Other uses:
 Metabolic defects where several genes are involved in the disease phenotype
 Detection of the actual protein/amino acid alterations

A

BIOCHEMICAL METHODS

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12
Q

WHAT ARE THE 5 BIOCHEMICAL METHODS

A
  1. Enzyme immunoassays 2. Immunohistochemistry 3. High-performance liquid chromatography (HPLC)
  2. Gas chromatography
  3. Mass spectrophotometry
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13
Q

 Detects the presence of metabolites in the blood, urine, or other biological fluids

A

ENZYME IMMUNOASSAYS

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14
Q

 Involve the use of specific antibodies or other ligands to detect the presence of the target molecules

A

ENZYME IMMUNOASSAYS

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15
Q

Widely used EIA

A

enzyme-linked immunosorbent assays (ELISA)

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16
Q

 Longstanding method that allows detection of protein abnormalities in situ

A

IMMUNOHISTOCHEMISTRY (IHC)

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17
Q

Formalin-fixed paraffin-embedded tissue:

A

<5 micron slices (microtome)

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18
Q

Fixation can affect tissue antigens altering/covering some epitopes  can be solved by antigen retrieval: enzyme digestion (proteinase K, chymotrypsin, pepsin, pronase) & heating tissue sections in water/buffer

A

Formalin-fixed paraffin-embedded tissue

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19
Q

Snap frozen tissue (in isopentane, at -160ºC):

A

5 to 15-micron slices (cryostat inside of a chamber held at 20ºC)

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20
Q

SNAP FROZEN TISSUE FIXATIVE

A

ACETONE

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21
Q

Sections are dried & stored frozen

A

SNAP FROZ2N TISSUE

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22
Q

Rehydration of the dried sections in of snap frozen tissue

A

phosphate buffered saline

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23
Q

2 Imaging/microscopic observation of antibody binding requires a signal from the antibody:

A

Fluorescent signal
Colorimetric signal

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24
Q

fluorescent molecules (fluorescein, Cy5, phycoerythrin)

A

Fluorescent signal

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25
Q

substrate solution is added, oxidized by the enzymes (horseradish peroxidase/alkaline phosphatase)  most frequently used: red/ brown IHC staining

A

Colorimetric signal

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26
Q

 Separation of molecules (NA & proteins) in solution through interaction with a solid support in the column

A

HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)

27
Q

2 PHASE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

A

mobile phase (solvent) & stationary phase (solid support)

28
Q

increase resolution & lower separation time while using less solvent; faster flow rates (5 mL/min)

A

Ultra-HPLC (UHPLC)

29
Q

Separation of vaporized sample through a column of inert carrier gas (mobile phase) & liquid (stationary phase) that differently adsorbs molecules

A

GAS CHROMATOGRAPHY (GC)

30
Q

DETECTOR OF GAS CHROMATOGRAPHY (GC)

A

flame ionization detector

31
Q

 Used for detection of drugs & poisons & their metabolites in biological samples
 May be coupled with MS to detect biomarkers of disease

A

GAS CHROMATOGRAPHY (GC)

32
Q

 Converts molecules to ions that can be moved in a magnetic field based on their charge & mass

A

MASS SPECTROMETRY
(MS)

33
Q

2 ionization methods for large biomolecules (proteins) of MASS SPECTROMETRY (MS)

A

a. Electrospray ionization (ESI)
b. Matrix-assisted laser desorption/ionization (MALDI)

34
Q

 High MW molecules
 Ionized molecules are accelerated at a fixed point & allowed to drift through the flight tube to the detector

A

MALDI-TOF (time-of-flight) spectrometry

35
Q

 Combined w/ TOF offers flexibility in the ID & quantification of peptides

A

Surface-enhanced laser desorption/ionization (SELDI)

36
Q

 Performed on a variety of specimen types: blood/buccal cells
 DNA mutations from single-base pair changes to large chromosomal rearrangements can be detected

A

NUCLEIC ACID ANALYSES

37
Q

simplified mutation detection (limiting specimens)

A

PCR amplification

38
Q

most definitive method for detecting mutations

A

DNA sequencing

39
Q

4 HYBRIDIZATION-BASED METHODS

A

Single-Strand Conformation Polymorphism (SSCP)
SEQUENCING (POLYMERIZATION) BASED METHODS
ENZYMATIC & CHEMICAL CLEAVAGE METHODS
OTHER METHODS

40
Q

 Based on the preference of DNA to exist in a double-stranded state
 Absence of the complementary strand: nucleic acids form intrastrand duplexes, 3D structure (conformer)
 Shape: kinks, loops, bubbles, tail

A

Single-Strand Conformation Polymorphism (SSCP)

41
Q

 Different from the normal sequence (control) conformers
 presence of gene mutation
 Detected by silver stain, radioactivity, or fluorescence

A

Bands/peak patterns

42
Q

 Utilizes differences in the Tm of short sequences (20 bases) w/ 1 or 2 mismatches & those w/ no mismatches
 Synthetic ss-probes (labeled) w/ normal/mutant target DNA sequence (immobilized) in a solution
 At specific annealing temperatures & conditions (stringency)
a. Probe will not bind to a near complementary target sequence w/ 1 or 2 mismatched bases
b. Probe w/ perfect complementary sequence, will bind

A

Allele-Specific Oligomer Hybridization
(ASO)

43
Q

 Method of analyzing the dissociation of dsDNA during the heating cycles
 PCR amplicons in the presence of a DNA-specific fluorescent dye (EtBr, SYBR green, LC green) are heated (0.3ºC/sec)
 Rise in the temperature, DNA duplexes begin to separate into single strands, losing the dye
 Black line
 targets w/ different mismatches to the hybridization probe

A

Melt Curve Analysis (MCA)

44
Q

overlaying peaks at expected Tm

A

Specimen w/ identical sequence

45
Q

2 or more peaks at different temperatures

A

Specimen w/ different sequence

46
Q

 High specificity  Uses fluorescent resonance energy transfer (FRET) probes
 Raise in temperature, probes dissociate at specific Tm  donor no longer close to the acceptor  fluorescence drops

A

High-resolution melt-curve analysis (HRMCA)

47
Q

Tm lower than that of the probe & its perfect complement, or sequence difference between probe reference sequence & the test sequence

A

High-resolution melt-curve analysis (HRMCA) Indication of mutation

48
Q

Formed when single strands that are not completely complementary hybridize to 1 another

A

Heteroduplexes

49
Q

Can be resolved through polyacrylamide/agarose gel electrophoresis:

A

presence of bands different from a homozygous reference control is indicative of mutation

50
Q

Test DNA is fragmented by treatment with DNase before binding to the complementary probes on the array

A

High-density oligonucleotide arrays

51
Q

2 TYPES OF Hybridization formats

A

Standard tiling
Redundant tiling

52
Q

base substitution is always in the 12th position from the 3’ end of the probe

A

Standard tiling

53
Q

same mutation is placed at different positions in the probe *Mutations are identified as indicated by which probes are bound

A

Redundant tiling

54
Q

uses sets of color-coded polystyrene beads in suspension as the solid matrix

A

Bead-array technology

55
Q

presence/absence of a mutation/polymorphism

A

Combination of bead color & test label

56
Q

 Detect point mutations & other SNPs  Primer 3’ end falls on the nucleotide to be analyzed
 Must match the template perfectly to be extended by Taq polymerase
 Presence/absence of the product = presence/absence of the mutation

A

Sequence-Specific (Primer) PCR (SSP-PCR)

57
Q

 Thermal cyclers w/ fluorescent detection
 RT-PCR, using 2 probes labeled 3’ quencher molecules & different fluors on the 5’ ends (complementary to either normal/mutant sequence)
 Presence of corresponding fluorescent signal indicates whether the test sequence is normal/mutant

A

Allelic Discrimination with
Fluorogenic Probes

58
Q

 Can detect sequence alterations  Mutation changes the structure of a restriction enzyme target site/changes the size of a fragment
 PCR-RFLP is used

A

Restriction Fragment Length
Polymorphisms (RFLPs)

59
Q

 Heteroduplex analysis using duplex RNA
 T7 or SP6 phage RNA polymerase  Detection of mutation: heteroduplexes form between normal & mutant
transcripts
 targets for cleavage by RNase enzymes (E. coli RNase & Aspergillus RNase T1)
 Remaining dsRNA fragments can then be separated by agarose gel electrophoresis

A

Nonisotopic RNase Cleavage Assay
(NIRCA)

60
Q

 Based on the characteristic enzymatic activity of cleavase
 Premixed reagents (including cleavase) + standard 96 well-plate + test specimens + controls

A

Cleavage Assay

61
Q

recognizes the structure formed by hybridization of the normal/mutant probes to the test sequences

A

Cleavase

62
Q

Combination of methods to increase sensitivity & detection (RFLP with modified primers)

A

other metjods

63
Q

Array-based methods & massive parallel sequencing methods provide specific multiplex detection & sensitivity required for clinical applications

A

other methods

MOLECULAR BIOLOGY (9 decks)

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